Role of cell polarity and planar cell polarity (PCP) proteins in spermatogenesis

Abstract

Studies on cell polarity proteins and planar cell polarity (PCP) proteins date back to almost 40?years ago in Drosophila and C. elegans when these proteins were shown to be crucial to support apico-basal polarity and also directional alignment of polarity cells across the plane of an epithelium during morphogenesis. In adult mammals, cell polarity and PCP are most notable in cochlear hair cells. However, the role of these two groups of proteins to support spermatogenesis was not explored until a decade earlier when several proteins that confer cell polarity and PCP proteins were identified in the rat testis. Since then, there are several reports appearing in the literature to examine the role of both cell polarity and PCP in supporting spermatogenesis. Herein, we provide an overview regarding the role of cell polarity and PCP proteins in the testis, evaluating these findings in light of studies in other mammalian epithelial cells/tissues. Our goal is to provide a timely evaluation of these findings, and provide some thought provoking remarks to guide future studies based on an evolving concept in the field.     

Basolateral protein Scribble binds phosphatase PP1 to establish a signaling network maintaining apicobasal polarity

Scribble, a member of the LAP protein family, contributes to the apicobasal polarity (ABP) of epithelial cells. The LAP-unique region of these proteins, which is essential and sufficient for ABP, includes a conserved Leucine-Rich Repeat (LRR) domain. The major binding partners of this region that could regulate ABP remain unknown. Here, using proteomics, native gel electrophoresis, and site-directed mutagenesis, we show that the concave surface of LRR domain in Scribble participates in three types of mutually exclusive interactions—(i) homodimerization, serving as an auto-inhibitory mechanism; (ii) interactions with a diverse set of polarity proteins, such as Llgl1, Llgl2, EPB41L2, and EPB41L5, which produce distinct multiprotein complexes; and (iii) a direct interaction with the protein phosphatase, PP1. Analogy with the complex between PP1 and LRR domain of SDS22, a well-studied PP1 regulator, suggests that the Scibble-PP1 complex stores a latent form of PP1 in the basolateral cell cortex. Such organization may generate a dynamic signaling network wherein PP1 could be dispatched from the complex with Scribble to particular protein ligands, achieving fast dephosphorylation kinetics.     

Apical spectrin is essential for epithelial morphogenesis but not apicobasal polarity in Drosophila.

Changes in cell shape and position drive morphogenesis in epithelia and depend on the polarized nature of its constituent cells. The spectrin-based membrane skeleton is thought to be a key player in the establishment and/or maintenance of cell shape and polarity. We report that apical beta(Heavy)-spectrin (beta(H)), a terminal web protein that is also associated with the zonula adherens, is essential for normal epithelial morphogenesis of the Drosophila follicle cell epithelium during oogenesis. Elimination of beta(H) by the karst mutation prevents apical constriction of the follicle cells during mid-oogenesis, and is accompanied by a gross breakup of the zonula adherens. We also report that the integrity of the migratory border cell cluster, a group of anterior follicle cells that delaminates from the follicle epithelium, is disrupted. Elimination of beta(H) prevents the stable recruitment of alpha-spectrin to the apical domain, but does not result in a loss of apicobasal polarity, as would be predicted from current models describing the role of spectrin in the establishment of cell polarity. These results demonstrate a direct role for apical (alphabeta(H))(2)-spectrin in epithelial morphogenesis driven by apical contraction, and suggest that apical and basolateral spectrin do not play identical roles in the generation of apicobasal polarity.     

Orchestrating stem cell fate: Novel tools for regenerative medicine

Mesenchymal stem cells are undifferentiated cells able to acquire different phenotypes under specific stimuli. In vitro manipulation of these cells is focused on understanding stem cell behavior, proliferation and pluripotency. Latest advances in the field of stem cells concern epigenetics and its role in maintaining self-renewal and differentiation capabilities. Chemical and physical stimuli can modulate cell commitment, acting on gene expression of Oct-4, Sox-2 and Nanog,the main stemness markers, and tissue-lineage specific genes. This activation or repression is related to the activity of chromatin-remodeling factors and epigenetic regulators, new targets of many cell therapies. The aim of this review is to afford a view of the current state of in vitro and in vivo stem cell applications,highlighting the strategies used to influence stem cell commitment for current and future cell therapies. Identifying the molecular mechanisms controlling stem cell fate could open up novel strategies for tissue repairing processes and other clinical applications.     

Orchestration of tissue‐scale mechanics and fate decisions by polarity signalling

Eukaryotic development relies on dynamic cell shape changes and segregation of fate determinants to achieve coordinated compartmentalization at larger scale. Studies in invertebrates have identified polarity programmes essential for morphogenesis; however, less is known about their contribution to adult tissue maintenance. While polarity‐dependent fate decisions in mammals utilize molecular machineries similar to invertebrates, the hierarchies and effectors can differ widely. Recent studies in epithelial systems disclosed an intriguing interplay of polarity proteins, adhesion molecules and mechanochemical pathways in tissue organization. Based on major advances in biophysics, genome editing, high‐resolution imaging and mathematical modelling, the cell polarity field has evolved to a remarkably multidisciplinary ground. Here, we review emerging concepts how polarity and cell fate are coupled, with emphasis on tissue‐scale mechanisms, mechanobiology and mammalian models. Recent findings on the role of polarity signalling for tissue mechanics, micro‐environmental functions and fate choices in health and disease will be summarized.     

GNrep mouse: A reporter mouse for front–rear cell polarity

Cell migration is essential during development, regeneration, homeostasis, and disease. Depending on the microenvironment, cells use different mechanisms to migrate. Yet, all modes of migration require the establishment of an intracellular front–rear polarity axis for directional movement. Although front–rear polarity can be easily identified in in vitro conditions, its assessment in vivo by live‐imaging is challenging due to tissue complexity and lack of reliable markers. Here, we describe a novel and unique double fluorescent reporter mouse line to study front–rear cell polarity in living tissues, called GNrep. This mouse line simultaneously labels Golgi complexes and nuclei allowing the assignment of a nucleus‐to‐Golgi axis to each cell, which functions as a readout for cell front–rear polarity. As a proof‐of‐principle, we validated the efficiency of the GNrep line using an endothelial‐specific Cre mouse line. We show that the GNrep labels the nucleus and the Golgi apparatus of endothelial cells with very high efficiency and high specificity. Importantly, the features of fluorescent intensity and localization for both mCherry and eGFP fluorescent intensity and localization allow automated segmentation and assignment of polarity vectors in complex tissues, making GNrep a great tool to study cell behavior in large‐scale automated analyses. Altogether, the GNrep mouse line, in combination with different Cre recombinase lines, is a novel and unique tool to study of front–rear polarity in mice, both in fixed tissues or in intravital live imaging. This new line will be instrumental to understand cell migration and polarity in development, homeostasis, and disease.     

Asymmetric cell division from a cell to cells: Shape,length, and location of polarity domain

Asymmetric cell division is one of the most elegant biological systems by which cells create daughter cells with different functions and increase cell diversity. In particular, PAR polarity in the cell membrane plays a critical role in regulating the whole process of asymmetric cell division. Numerous studies have been conducted to determine the underlying mechanism of PAR polarity formation using both experimental and theoretical approaches in the last 10 years. However, they have mostly focused on answering the fundamental question of how this exclusive polarity is established but the precise dynamics of polarity domain have been little notified. In this review, I focused on studies on the shape, length, and location of PAR polarity from a theoretical perspective that may be important for an integrated understanding of the entire process of asymmetric cell division.     

Limited expression of nuclear pore membrane glycoprotein 210 in cell lines and tissues suggests cell-type specific nuclear pores in metazoans

The nuclear pore complex (NPC) is the only known gateway for nucleocytoplasmic traffic. The nuclear pore membrane glycoprotein 210 (POM210/gp210) is considered to be important for the assembly and structure of pore complexes in metazoan cells. However, here we demonstrate cell-type specific expression of the gp210 protein during mouse organogenesis. As shown previously for its mRNA, distinct expression of the gp210 was seen in developing epithelia and some other cell types, whereas it was undetectable in nuclei of several other embryonic tissue compartments. In sharp contrast, monoclonal antibody 414 recognizing four non-membrane nucleoporins, stained the nuclear envelope of all cell types. In four cultured mouse cell lines, gp210 mRNA and protein were below detection levels, in contrast to some other nucleoporins tested. Distinct expression of gp210 mRNA and protein was seen in cultured mouse embryonic stem (ES) cells. These findings support the view of cell-type specific NPCs in metazoans and that the gp210 gene is regulated by cell-type specific control elements not shared by other nucleoporins. Although it cannot be excluded that very low expression levels of gp210 are sufficient to allow attachment of NPCs, a more likely alternative is that it has cell-type specific functions.     

The autonomous cell fate specification of basal cell lineage: the initial round of cell fate specification occurs at the two‐celled proembryo stage

In angiosperms, the first zygotic division usually gives rise to two daughter cells with distinct morphologies and developmental fates, which is critical for embryo pattern formation; however, it is still unclear when and how these distinct cell fates are specified, and whether the cell specification is related to cytoplasmic localization or polarity. Here, we demonstrated that when isolated from both maternal tissues and the apical cell, a single basal cell could only develop into a typical suspensor, but never into an embryo in vitro. Morphological, cytological and gene expression analyses confirmed that the resulting suspensor in vitro is highly similar to its undisturbed in vivo counterpart. We also demonstrated that the isolated apical cell could develop into a small globular embryo, both in vivo and in vitro, after artificial dysfunction of the basal cell; however, these growing apical cell lineages could never generate a new suspensor. These findings suggest that the initial round of cell fate specification occurs at the two‐celled proembryo stage, and that the basal cell lineage is autonomously specified towards the suspensor, implying a polar distribution of cytoplasmic contents in the zygote. The cell fate transition of the basal cell lineage to the embryo in vivo is actually a conditional cell specification process, depending on the developmental signals from both the apical cell lineage and maternal tissues connected to the basal cell lineage.     

Hippo kinases maintain polarity during directional cell migration in Caenorhabditis elegans

Precise positioning of cells is crucial for metazoan development. Despite immense progress in the elucidation of the attractive cues of cell migration, the repulsive mechanisms that prevent the formation of secondary leading edges remain less investigated. Here, we demonstrate that Caenorhabditis elegans Hippo kinases promote cell migration along the anterior–posterior body axis via the inhibition of dorsal–ventral (DV) migration. Ectopic DV polarization was also demonstrated in gain‐of‐function mutant animals for C. elegans RhoG MIG‐2. We identified serine 139 of MIG‐2 as a novel conserved Hippo kinase phosphorylation site and demonstrated that purified Hippo kinases directly phosphorylate MIG‐2^S139. Live imaging analysis of genome‐edited animals indicates that MIG‐2^S139 phosphorylation impedes actin assembly in migrating cells. Intriguingly, Hippo kinases are excluded from the leading edge in wild‐type cells, while MIG‐2 loss induces uniform distribution of Hippo kinases. We provide evidence that Hippo kinases inhibit RhoG activity locally and are in turn restricted to the cell body by RhoG‐mediated polarization. Therefore, we propose that the Hippo–RhoG feedback regulation maintains cell polarity during directional cell motility.     

Applications of single cell RNA sequencing to research of stem cells

Stem cells(SCs) with their self-renewal and pluripotent differentiation potential,show great promise for therapeutic applications to some refractory diseases such as stroke, Parkinsonism, myocardial infarction, and diabetes. Furthermore, as seed cells in tissue engineering, SCs have been applied widely to tissue and organ regeneration. However, previous studies have shown that SCs are heterogeneous and consist of many cell subpopulations. Owing to this heterogeneity of cell states, gene expression is highly diverse between cells even within a single tissue,making precise identification and analysis of biological properties difficult, which hinders their further research and applications. Therefore, a defined understanding of the heterogeneity is a key to research of SCs. Traditional ensemble-based sequencing approaches, such as microarrays, reflect an average of expression levels across a large population, which overlook unique biological behaviors of individual cells, conceal cell-to-cell variations, and cannot understand the heterogeneity of SCs radically. The development of high throughput single cell RNA sequencing(scRNA-seq) has provided a new research tool in biology, ranging from identification of novel cell types and exploration of cell markers to the analysis of gene expression and predicating developmental trajectories. scRNA-seq has profoundly changed our understanding of a series of biological phenomena. Currently, it has been used in research of SCs in many fields, particularly for the research of heterogeneity and cell subpopulations in early embryonic development. In this review, we focus on the scRNA-seq technique and its applications to research of SCs.     

A protein interaction map for cell polarity development

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.     

Role of the cellular environment in interstitial stem cell proliferation in Hydra

Summary The role of the cellular environment on hydra stem cell proliferation and differentiation was investigated by introduction of interstitial cells into host tissue of defined cellular composition. In epithelial tissue lacking all non-epithelial cells the interstitial cell population did not grow but differentiated into nerve cells and nematocytes. In host tissue with progressively increased numbers of nerve cells growth of the interstitial cell population was positively correlated to the nerve cell density. In agreement with previous observations (Bode et al. 1976), growth of the interstitial cell population was also found to be negatively correlated to the level of interstitial cells present. The strong correlation between the growth of the interstitial cell population and the presence of interstitial cells and nerve cells implies that interstitial cell proliferation is controlled by a feedback signal from interstitial cells and their derivatives. Our results suggest that the cellular environment of interstitial cells provides cues which are instrumental in stem cell decision making. Offprint requests to: T.C.G. Bosch